Method and apparatus for maldi analysis

ABSTRACT

Matrix assisted laser desorption/ionization is performed in a manner to thermalize large analyte ions in a plume of desorbed material for spectroscopic analysis. The thermalized ions have a low or zero mean velocity and are presented at a well-defined instant in time, reducing artifacts and sharpening the spectral peaks. In one embodiment the light is delivered to a matrix or sample holder having a cover, baffle or compartment. The baffle or compartment impedes or contains a plume of desorbed material and the analyte undergoes collisions to lower its mean velocity and directionality. Thus “thermalized” the analyte ions are passed to a mass analysis instrument. In a preferred embodiment an optical fiber butts up against a thin transparent plate on which the specimen resides, with the matrix side in a vacuum acceleration chamber. A mechanical stage moves the specimen in both the x- and y- directions to select a point on the specimen which is to receive the radiation. The use of a fiber optic illuminator allows the entire stage assembly to be subsumed essentially within the dimensions of a conventional stage. In other embodiments, a thermalizing compartment is provided in a capillary tube about the end of the illumination fiber and the sample matrix is deposited along the inner cylindrical wall of the tube, so the capillary forms a migration path to the outlet for thermalization of the desorbed analyte. In other embodiments microstructures having the shape of a small lean-to, overhang or perforated cover plate, or providing a high aspect surface texture, provide the necessary containment to promote thermalization of the released analyte. A thin layer or cover of fibrous or permeable material may also be used to thermalize the analyte before mass analysis, and in other embodiment this material may also act as the substrate. An automated instrument may include a fixed array of illumination fibers which are illuminated at different times to eject samples from a corresponding array of points on the specimen.

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No.08/934,455, filed Sep. 19, 1997, which is a continuation-in-part of U.S.application Ser. No. 08/710,565, filed Sep. 19, 1996, now U.S. Pat. No.5,777,324, each of which is incorporated herein by reference in itsentirety.

BACKGROUND

[0002] The present invention relates to Matrix Assisted LaserDesorption/Ionization Mass Spectrometry (MALDI-MS). This methodintroduced by Karas and Hillenkamp in 1988 has become established as amethod for mass determination of biopolymers and substances such aspeptides, proteins and DNA fragments. In this method, the substance tobe analyzed is typically placed in a solution of matrix material andcoated onto a support. The solute evaporates, leaving the analyte in asolid matrix which is then illuminated to cause the analyte molecules orsynthetic polymers to be desorbed. This desorption process is especiallyuseful for releasing large biological molecules without charring,fragmentation or chemical degradation to a mass spectrometer or similarinstrument for separation and detection.

[0003] In common MALDI mass spectrometry setups, the sample is placed ona metal substrate and is irradiated from the side facing the ionanalyzer (“top illumination”). In contrast to this arrangement,transmission illumination is also possible wherein the rear side of thesample is illuminated through a transparent substrate, for example by afocused laser beam. In either event, the matrix consisting of arelatively low molecular weight aromatic organic substance such asnicotinic acid, benzoic or cinnamic acid, holds a quantity of thepeptide, protein or other substance to be analyzed and is irradiatedwith a high intensity laser beam to cause desorption of the analyte froma surface of the sample. In a representative mass spectrometryarrangement, ions of the analyte are subjected to electric fieldacceleration for mass separation in the instrument, such as atime-of-flight (TOF) mass spectrometer. The laser radiation is selectedsuch that matrix strongly absorbs at the laser wavelength and desorbsmaterial sufficiently quickly to form an ejecting plume at its outersurface. The laser wavelength, however, is sufficiently long to notbreak bonds of the analyte, and the desorption process is non-thermal sothat large molecules are provided to the spectrometer instrumentsubstantially intact. The mechanism of desorbing or ejecting largemolecules from a relatively light matrix material is quite general, andallows one to detect analytes having a mass of tens or hundreds ofkiloDaltons. Various techniques for preparing certain classes ofsubstances for desorption from a variety of matrix materials haveextended the usefulness of the technique.

[0004] In conventional instruments, the standard configuration involvesperforming both illumination and mass analysis from the same side of thesample. This produces a relatively high yield of large molecule ions andgood mass resolution. Illumination spot sizes of between 50 and 1000micrometers in diameter have been used with illumination levels in therange of 10⁶-10⁷ watts/square centimeter to essentially eject smallvolumes of the sample and provide a short, high velocity plume ofmaterial for analysis. An extensive library of organic large moleculespectra has been built up using such instruments. Nonetheless, thegeometrical requirements of providing an ion extraction and accelerationoptics in a vacuum flight chamber with a number of high voltageelectrodes to accelerate the material to an analysis detector imposeconstraints on the optical path of the laser illumination beam,resulting in a relatively costly and inflexible instrument. Furthermore,the provision of a relatively high energy plume as the initial ionsource results in a spread of velocities and spatial position of theinitial burst of ions, which in addition are subject to differingelectric fields because of the spatial spread of the plume, so that,while the instrument provides a good yield of analyte, the massresolution is compromised. In general, the matrix molecules provide aninternal reference peak. However, since the analyte is often many timesgreater in mass, and the mechanism of desorption is also not fullyunderstood, it is also possible that the acquired spectra includeunrecognized shifts and other artifacts resulting from initial plumegeometry or release dynamics that will complicate the accuracy of massdetermination and a comparison with independently produced spectra inthe future.

[0005] Various researchers have explored transmission MALDI fordifferent materials and one or more matrix compositions, and have beenable under some conditions to obtain results analogous to thoseaccumulated using the more prevalent top side illumination. In general,by separating illumination and the mass analysis instrumentation onopposite sides of a matrix one might expect to implement differentinstrument designs with greater freedom. In particular, scanningarrangements might be implemented to allow the selective analysis ofparticular spots or sample locations. Furthermore, the ability toprovide ion desorption from a defined surface may be expected to yieldsharper spectra. However in switching from a top illuminationconfiguration to a transmission illumination configuration, one ofnecessity changes the nature of a number of essential processesunderlying the desorption technique. Thus for example the shape of theplume, the velocities or directions of molecules or ions upon release,and the underlying mechanism or yield of the release may all be affectedby a change in the illumination/desorption geometry. Relatively fewexperiments have utilized transmission illumination, and these have ingeneral yielded lower quality spectra than top illumination, and havebeen tried only with a limited range of relatively light analytemolecules.

[0006] The essential mechanisms by which material is desorbed are notfully understood, and effects may vary with different materials. Ingeneral, to make a measurement one tunes the analyzer by settingappropriate electric and/or magnetic fields, or otherwise defining asample window, then illuminates the matrix, progressively increasing thefluence until the spectrometer starts to detect desorbed ions. Thefluence may generally then be increased somewhat to increase the amountof the heavy analyte present in the desorbed material, but should not beincreased so much as to introduce charring or fragmentation of thematerial. In general, increase in illumination fluence increases theamount of material released. However, as described above, the massresolution, which is initially limited, may suffer due to an increasedspread of initial velocities, the irregular geometry of the emissionplume or other factors.

[0007] It is therefore desirable to provide a transmission MALDI methodor apparatus in which resulting spectra are identical to or wellcorrelated with MALDI spectra obtained in top illumination of similarcompounds.

[0008] It is also desirable to provide new transmission MALDI stages ormechanisms for desorption of an analysis sample.

[0009] It is also desirable to provide a MALDI spectrometry processwherein the peaks are improved, and mass resolution refined.

SUMMARY

[0010] One or more of the above desirable ends are achieved inaccordance with the present invention by carrying out matrix assistedlaser desorption analysis in a manner to inject analyte ions into aspectrometric analysis system with a low or zero mean velocity at adefined instant in time. In one embodiment the laser light is deliveredto a matrix or sample holder having a cover, baffle or compartment. Thelaser illuminates the matrix, preferably over a relatively large spot ata fluence in the range of 10⁶ watts/square centimeter, causing thedesorption of analyte and matrix material which is released in a plume.The baffle or compartment impedes or contains the plume toinstantaneously define a region with a relatively high density ofanalyte and matrix. The analyte, which may have a molecular weight tensto thousands of times greater than the matrix, undergoes collisions toachieve a mean velocity which is low or zero. Thus “thermalized” theanalyte ions are passed from the baffle region to undergo a conventionalmass analysis.

[0011] In a preferred embodiment of a stage useful for the practice ofthe invention, the laser illumination is provided by the output end ofan optical fiber which may for example receive illumination at its inputend from a gas laser, a hybrid frequency-multiplying laser arrangement,a high powered laser diode array, or a diode-pumped source. The end ofthe optical fiber butts up against a thin transparent plate on which thesample reside, the sample being on the side of the plate opposite thefiber facing in a vacuum acceleration chamber. Preferably, a mechanicalstage moves the plate in both the x- and y- in-plane directions toselect a sample or a point on a given sample which is to receive theradiation. The use of a fiber optic illuminator allows the entire stageassembly to be subsumed essentially within the dimensions of aconventional instrument stage yet provide a robust and accurateillumination source of well defined intensity and high uniformity.Emission from the source then illuminates the sample, causing theanalyte to be desorbed at a surface of the plate facing the massanalyzer assembly. In accordance with one aspect of the invention theplume is partially confined so that its highly directional momentum israndomized, or “thermalized”. After thermalizing, the plume environmentcontains slow ions which are accelerated in the analyzer, separatingeach characteristic charge/mass component into a sharply definedinterval for spectrometric detection.

[0012] In other embodiments, a thermalizing region is defined by a smallferrule or capillary-like tube enclosure which surrounds a region at theend of the illumination fiber. The matrix is deposited along the innercylindrical wall of the tube, where the divergent fiber outputilluminates the matrix. The tube provides a short tunnel as a migrationpath to the outlet in which the desorbed material is initially ejectedwith oblique paths for thermalization of the desorbed analyte. In otherembodiments microstructures having the shape of a small lean-to,overhang or perforated cover plate provide containment to increaseresidence time or provide collisional interaction for thermalization ofthe released analyte. Such a confining microstructure can also be formedby the sample crystals and the surface of the substrate if thecrystallization process is specifically controlled to achieve suchstructures.

[0013] The latter constructions may include a two stage releaseconfiguration wherein the laser illumination forms a plume which thenfills a compartment. The compartment has an opening in one wall throughwhich the thermalized ions which have migrated from the illuminationplume are emitted. In this two-stage construction, the distance betweenillumination and outlet is made large enough to thermalize the largemolecules, but small enough to assure that emission of analyte ionsoccurs in a short time interval that does not broaden the TOF peakwidth.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] These and other features of the invention will be understood fromthe description of representative embodiments of the method herein andthe disclosure of illustrative apparatus for carrying out the method,taken together with the Figures, wherein

[0015]FIG. 1 illustrates a prior art MALDI analysis technique;

[0016]FIG. 2 illustrates steps of MALDI analysis in accordance with thepresent invention;

[0017]FIGS. 3A and 3B illustrate a basic embodiment of an apparatus inaccordance with the present invention for two different sample mountsfor a transmission MALDI analysis, respectively;

[0018]FIGS. 4A and 4B illustrate stages of operation of the embodimentof FIGS. 3A or 3B;

[0019]FIGS. 5A and 5B illustrate a second embodiment and its operation;

[0020]FIGS. 6A and 6B illustrate a third embodiment and its operationfor top illumination;

[0021]FIGS. 7 and 7A illustrate an experimental MALDI set up;

[0022]FIGS. 8A and 8B show a preferred stage for practice of theinvention;

[0023]FIGS. 9A and 9B illustrate a fourth embodiment of the invention;

[0024]FIG. 10 schematically depicts a pin tool apparatus;

[0025]FIG. 11 depicts various pin conformations. FIGS. 11A shows a solidpin with a straight head. FIG. 11B shows a solid pin with a concavehead. FIG. 11C shows a solid pin with a truncated pyramidal head. FIG.11D shows a pin with a concave head and a hollowed center (through whichcan be inserted an optical fibre). FIG. 11E shows a pin with a truncatedpyramidal head and hollowed center.

[0026] FIGS. 12A-C schematically represent a pintool apparatus andmount, each separately and a cross section of the mount and toolinstalled.

[0027]FIG. 13 and FIG. 13A show a schematic representation of massspectrometry geometries for the pintools shown in FIG. 10, FIGS. 11A-E,and FIG. 12B.

[0028]FIG. 14 schematically depicts a pintool onto which a voltage isapplied. When an electrical field is applied, nucleic acids areattracted to the anode. This system would also purify nucleic acids,since uncharged molecules would remain in solution, while positivelycharged molecules would be attracted towards the cathode.

[0029]FIG. 15 shows a flow chart of the steps involved in sequencing bymass spectrometry using post-biology capture.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030]FIG. 1 shows a representative prior art set up for the matrixassisted laser desorption and ionization of a specimen such as a largemolecule compound having a molecular weight, for example, in the rangeof 500 to 100,000 daltons or more. As shown, a substrate 3 such as asheet of metal foil or a glass slide, bears a sample 2 in a region onits surface. As discussed above, sample 2 is generally deposited as asolution of a relatively low molecular weight laser-absorbent materialwith a minor amount of the large molecule analyte contained therein, andit is allowed to dry in place to form a thin layer of solid materialwhich may for example consist of a granular or continuous bed ofcrystals. In special cases a solvent, stable in vaccuo is used such asglycerol. The sample 2 then forms a thin liquid layer or droplet onsubstrate 3. A laser beam 1 is directed at the sample 2 causing a plume4 of material to be desorbed therefrom. In general, the matrix materialis taken to be a low molecular weight compound, e.g. an aromaticcompound for desorption with wavelengths in the ultraviolet (UV), andthe laser has a wavelength selected to excite and be strongly absorbedby the matrix material. Illumination of the sample then results indesorption of material therefrom and formation of a plume 4 whichexpands away from the illuminated site as shown in this figure. Whilethe mechanism of large molecule desorption is not fully understood, itis clearly different from a “thermal” evaporation and it is a very fastevent; the analyte therefore enters the surrounding vacuum chamberwithout extensive chemical degradation. However, as further illustratedin FIG. 1 the desorbed material is highly directional, having a largecomponent of velocity in the direction normal to the surface indicatedby vector V₁ in the figure. The plume expansion velocity is typically onthe order of 500 to 1,000 meters per second.

[0031] In a typical analysis process, the desorbed material in plume 4is provided to a mass spectrometer such as a sector or quadrupolescanning spectrometer which provides both acceleration and amass-dependent transmission from, or to a time of flight (TOF) massanalyzer. Generally a time of flight instrument is preferred since therelatively small amount of sample in the plume results in a very lowcollection efficiency, poor sensitivity and difficulty in setting up fora sector scanning instrument.

[0032] For a time of flight instrument, the kinetics of moleculartransport may be roughly modeled based on certain assumptions about thenature of the plume, the geometry of the plume generation stage, andsurrounding electrodes and field strengths. When setting up such amodel, the mass resolution, m/Δm, may, with the initial velocity andcertain simplifying assumptions, be approximated as the magnitude of asum of terms, including a term varying with the initial velocity spreadand plume extent in relation to electrode spacing. Basically, initialvelocity (energy) spread, the extent to which the plume subjectsmolecules to different field conditions, and the extent to which theions undergo collisions or experience a turnaround time in thedesorption and acceleration geometry, all introduce a spreading in thetime of arrival peaks at the detector.

[0033] These problems are addressed in accordance with a principalaspect of the present invention by a method for the laser desorption andionization of a sample as set forth in FIG. 2. In general, the sampleconsists of a solid matrix including the analyte material to bemeasured, and is deposited on a support for illumination by a laser,substantially as in the prior art. As set forth in FIG. 2 as a firststep the sample is illuminated causing material to be desorbedtherefrom. In general the illumination step is effected by a short pulseof laser radiation having a duration of 0.5-200 nanoseconds and directedat an illumination spot with a diameter between approximately tenmicrometers and several hundred micrometers, and an irradiance in therange of 10⁶ watts/square centimeter or more to form a plume of desorbedand ionized material. As noted above, this desorbed material initiallyhas a directional momentum imparted to it by the physical processes oflaser-induced desorption. In accordance with the present invention asecond step confines this desorbed material for a period such that thedirectional momentum is at least partially thermalized, i.e., randomizedin direction and reduced in overall magnitude, by undergoing internalcollisions and collisions with the surroundings before being subjectedto spectrometric separation. It is understood that the plume itself isof small dimension, and the confinement is of corresponding dimension,for example, with a wall spacing such that analyte molecules in theplume undergo at least one collision with a surrounding wall orobstruction. The invention also contemplates that the thermalization maybe accomplished using a two stage ion extraction in which low fieldstrength is provided in an initial region to result in an extendedresidence time near the initial site of the plume before acceleration.

[0034] In general the invention may be practiced with eithertransmission or, top illumination geometries, although different factorsenter into consideration for positioning the actual containment andacceleration structures in relation to the substrate. Representativephysical embodiments of the invention will be described below, withparticular reference to a time of flight mass analysis instrument.However as will be apparent from the general description, the inventionserves equally well and provides significant improvements when operatedas the sample input for various other analysis instruments such as anion trap, and ion cyclotron resonance Fourier transform massspectrometer, and other instruments used to detect or characterize ionsat low pressure.

[0035]FIGS. 3A and 3B illustrate a basic embodiment of the presentinvention, with the elements corresponding to those of FIG. 1 numberedcorrespondingly. As shown in FIG. 3A, an optical fiber 10 which may, forexample, have a core of 140 or 200 micrometers in diameter, is butted upagainst one face of a transparent substrate 3 upon which a sample 2comprised of a matrix with the analyte therein is deposited. Here adried matrix is illustrated. The fiber 10 illuminates the sample 2 withits divergent output beam 1 causing a plume of desorbed material toemanate therefrom. As further shown in FIG. 3A, a containment structure20 forms a compartment 21 about the plume having one or more exitapertures 22 in a wall thereof. The dimensions of the compartment 21 arecomparable to that of the illumination spot e.g. 10 to 500 micrometers,and may be of the same order of magnitude of the extent of plume, suchthat the plume expands to fill the compartment within a few hundrednanoseconds.

[0036] The configuration shown in FIGS. 3A and 3B are both transmissionillumination configurations in that the laser light illuminates thesample from a direction opposite to the ion extraction direction. In theconfiguration of FIG. 3A the sample is placed directly on thetransparent substrate such that the fiber illuminates the sample fromthe bottom side, i.e. the side facing the substrate. As a result thematerial is desorbed from the bottom side of the sample; the sample 2may for example consist of microcrystals in a layer several micrometersthick on a substrate and the plume may consist largely of material whichhas been desorbed from the bottom (substrate-facing) side of thecrystals and which has turned around before forming the main body of theprojecting plume.

[0037]FIG. 3B shows a corresponding configuration of this embodiment forreflection MALDI. In this embodiment, the sample 2 is deposited not onthe substrate 3 separating the fiber and analysis chamber, but on a wall20 of the enclosure. The fiber 10 then shines its beam through thesubstrate and across a small gap constituting the compartment to thesample on the far side. In either case, the plume expands into thecompartmentalized space 21.

[0038]FIGS. 4A and 4B illustrate the subsequent evolution of the plumeover time. As discussed above, the velocity of molecules in the plumemay initially be on the order of several hundred meters per second ormore. As shown in FIG. 4A, the plume evolves to fill the compartment 21and in general to undergo internal collisions among matrix molecules andbetween matrix and analyte molecules as well as with the walls 20 of thespecimen holder. This results in a randomization of the directions ofmovement of the molecules, and in the case of molecules having variousdegrees of freedom beyond the three orthogonal translational modes, asis typically the case for proteins and large organic molecules, theenergy may also be partitioned internally to result in a substantiallylower average velocity with a well behaved Maxwellian distribution. Asshown in FIG. 4A, the molecules reach the exit apertures 22 after arelatively short diffusion time. FIG. 4B then shows the plume materialaccelerated from the apertures upon the application of an electricextraction field E_(ext). Thus the apertures 22 provide the samplethrough discrete openings of defined size with a velocity which has beenat least partly randomized, and preferably fully randomized with aMaxwellian distribution and a low mean velocity. This defined initialcondition results in time of flight spectra having more sharply definedpeaks and less spreading, resulting in a calculated mass resolutionwhich is not only several times higher than conventional instruments,but can be independent of mass, resulting in a substantial improvementin spectral definition at high molecular weights.

[0039]FIGS. 5A and 5B illustrate a second embodiment 30 of the presentinvention in which a single body of novel construction has replaced thesubstrate and compartment of the first embodiment. Here, a high aspectcapillary 32 is held by an electrically conducting housing or jacket 9,which serves for example to provide a uniform and field flatteningconductive starting plane for the spectrometric acceleration. The sample2 is deposited on inside walls of the capillary, which in turn surroundsthe end of fiber 10 so that the output beam diverging from the fiber endirradiates the sample 2 and causes a plume of material to be ejectedfrom the sides of the capillary into the central lumen 33. In this case,if the sample 2 is coated as a band around the interior of thecapillary, the resultant plume is self-interfering, i.e. the highlydirectional desorption plume is aimed radially inward so that materialfrom different portions of the wall collides with plume material fromother portions resulting in a high incidence of collision within thesmall capillary volume 33. As further shown in FIG. 5B, the extractionelectric field results in acceleration of a thermalized sample ofmaterial 35 along a direction transverse to the capillary axis into theanalysis instrumentation.

[0040] The confinement geometry shown in FIG. 5A can also be used in atop illumination arrangement. In that case the laser beam emerging fromthe fiber 10 is preferably replaced by a focused or small diameter laserbeam entering the confinement from the front (i.e. facing the massanalyzer) side.

[0041] As is apparent from the foregoing example, the confinement of theplume need not amount to a total enclosure, but need only include anarrangement which assures collisional interaction of the desorbedmaterial, preferably including at least one collision with a wall of aphysical confining structure, which may for example change the directionof molecular travel and thus more quickly reduce the highly directionalmomentum by collisions among molecules of the desorbed material.

[0042]FIGS. 6A and 6B illustrate another confinement arrangement, whichin this case is configured for top side (i.e. the side of ionextraction) illumination. As before the numerals 1, 2, 3 and 4 designaterespectively the laser illumination beam, the sample, the substrateholding the sample and the plume emitted by desorption from the sample.In this embodiment, a structure 40 provides a barrier or overhang 42across the emission path of the plume. Overhang 42 may for exampleconsist of a shelf or cap held by a support or post 41. Overhang 42 doesnot entirely cover the sample 2, but extends like a lean-to with an opensector 43 allowing oblique illumination by the laser beam 1, focused toa small spot on the sample. As shown in FIG. 6B the plume thenthermalizes under the lean to and is accelerated out through the sector43, to provide slow ions to the analysis instrument, which traps oraccelerates them from that point outward.

[0043] In all embodiments for a plume thermalization such as theexamples shown in FIGS. 3 to 6, the ion extraction may be accomplishedby implementing several retarding and acceleration electric fieldsgenerated by suitable electrodes and/or meshes in the space between theplume confinement structures and the mass analyzer. One such mesh 80 ata potential U relative to the confinement is drawn in FIG. 3A by way ofillustration. All potentials of the retarding or extraction electrodesmay be time modulated, e.g. time delayed.

[0044] While the foregoing arrangements have shown physical barriers inwhich an external structure such as a cap, cover or enclosure providesthe thermalizing container, the invention further contemplates that thecontainment may result from more open structures implemented in part bythe matrix itself. FIGS. 7 and 7A illustrate an experimental set upwherein thermalization has been observed to occur in a transmission modeillumination geometry due to partially thermalizing conditions providedby a bed of microcrystals of matrix material. In this apparatus 50, alaser source was set up to provide illumination to a first fiber F₁ fortop illumination of the sample, or a second fiber F₂ for transmissionillumination from the backside of the sample. The sample was held on asubstrate 3 with the fiber F₂ carried by a stage with its end facepositioned exactly on the axis of the mass analyzer. The emerging laserbeam aims at a selected position on the sample which was controlled bythe stage carriage. As best seen in FIG. 7A, the sample 2 consisted of arelatively thin bed of microcrystals having a maximum size in the rangeof a few to a few hundred micrometers deposited on the surface of thesubstrate facing the analysis chamber. In this case it was found that asubstantial portion of the plume was emitted from the rear face of thecrystals, i.e. the crystal faces the substrate and fiber, across a gapof no more than 5 micrometers, so that the emitted material quicklycollided with the substrate and turned around to interact with plumematerial and result in a partially thermalized state of the desorbedmaterial reaching the analysis instrument. The microcrystals lay in arandom geometry, with rear faces lying a varying oblique angles so thatthe overall desorption momentum was initially less directional than fora top-illumination set-up, and quickly randomized further as themolecules collided and turned back into the flux of desorbed plumematerial.

[0045] As further shown in FIG. 7A, the stage 60 includes a metal plate61 which provided a substantially flat field about the substrate 3 forenhanced emission and acceleration geometry. The illumination fiber F₂passes through a slot in this planar electrode, and butts against thesubstrate 3. The fiber had a high numerical aperture and provided asubstantially homogeneous beam profile of defined diameter at thesample. In the illustrated embodiment, a 141 μm fiber was used such thatthe butt coupled geometry provided a 200 micrometer spot on the sample.As further shown in FIG. 7A, the sample consisted of a typical dryingdeposit of microcrystals and macrocrystals on a simple glass coversheet. The larger crystals were approximately 100 μm in dimension whilethe remaining microcrystals were much smaller and were completelyremoved by the initial actuations of the laser. The macrocrystalsthemselves naturally oriented with the substrate faces at an assortmentof angles so that the plume emitted at substantially normal incidencefrom these surfaces would quickly collide with the substrate, headingoff in a direction generally away from the substrate and back into theplume material, quickly undergoing thermalization.

[0046] Skipping ahead briefly to FIGS. 9A and 9B, there is shown yet afourth embodiment 70 of the invention, wherein containment, or moreaccurately, enhanced collisional interaction is provided by a porous orpermeable structure. In this embodiment 70, the sample 2 is placed on asubstrate 3, and covered with a shroud or sheath of porous or fibrousmaterial 71 mounted above the sample with a suitable spacer 72.Thermalization of the plume then occurs upon extraction of the ionsthrough the porous or fibrous sheath, with or without an appliedelectric field which may be provided by suitable electrodes. In atypical set up, the sheath 71 is thin, for example 100 μm or less, andthe spacer 72 provides a free distance of the order of the samplethickness. By way of example, a pulp-based fibrous paper similar to alaboratory filter paper was found to thermalize the emitted plume,although in general the material for this layer will be selected toneither react with nor contaminate the plume.

[0047] In the embodiment shown in FIG. 9A, the sample is illuminatedthrough the optically transparent substrate 3 in transmission geometryby a fiber 10 as described above. In the embodiment shown in FIG. 9B thesample is illuminated by a focused collimated light beam 1 in topillumination geometry through the porous or fibrous sheath 71 which isoptically transparent or translucent. Suitable materials for the fibrousor porous sheath 71 include glass, ceramic and polymeric materials. Aglass frit of a polymer or paper micropore membrane as used for cleaningor purifying liquids such as water is also suitable for the sheath 71.In an alternative embodiment (not illustrated) the sample can be placeddirectly onto the bottom side of the porous material, i.e., the sidefacing away from the mass analyzer. In another variation of thisembodiment, the sample is incorporated into an optically transparent ortranslucent sheath of the porous material, and is illuminated in situ sothat desorption occurs directly into the collision-enhancing porousstructure of the sheath.

[0048] The foregoing examples show thermalization with variousstructures and bodies to illustrate a range of implementations of theinvention, and these have involved simple mechanical elements. Howeverit is not necessary to build up the thermalizing plume stage out ofexisting structures such a screens or capillary tubes. It is alsopossible to devise completely new structures for holding a sample tocause emission into a localized thermalizing environment. Thus forexample the substrate 2 may consist of a plate having a plurality ofthin needle-like projections or deep wells at its instrument-facingsurface onto which the sample 2 is coated. In this case, theillumination 1 provided by the laser causes desorption from the sides ofthe pins, or from the walls of the wells, into a space confined by thesurrounding walls or adjacent pins. The initial plume velocity then issubstantially parallel to the general plane of the substrate and resultsin containment with a high collisional incidence, and subsequentemission of thermalized plume material across a plane normal thereto, atthe ends of the pins or wells formed on the substrate. Such pins orwells may be formed as microstructures by known techniques ofmicrolithography using compatible materials, and may for example beconfigured to additionally act as sample combs (e.g., hedgehog combs) ormicrotiter plates, allowing the support from a previous preparationprocess to serve as the substrate in a MALDI analyzer. Other novelmicrolithographic constructions will be readily implemented to providean effective collision space for modulating the plume. Thermalizationmay be further improved by provision of a “collision buffer gas” to theconfined, desorbed material. Preferably, the collision buffer gaspromotes collisional dissociation, chemical ionization and/ormolecule-molecule or ion-molecule reaction with the analyte.

[0049] A preferred embodiment of the invention is implemented with thesubstrate 3 carried on a stage 12 which is configured in conjunctionwith the fiber carrier to allow the fiber to illuminate any selected x,y position on the substrate 3 and desorb material therefrom. Such astage is illustrated in FIGS. 8A and 8B. As shown in perspective view inFIG. 8A, the fiber 10 is mounted in a collar 10 a fixed to a plate 11which provides a stable mechanical mounting to position the end of thefiber protruding from plate 11 into an opening 12 a in a stage plate 12.The fiber end face is exactly centered in a fixed position on the axisof the mass analyzer so as to determine a symmetrical distribution ofions injected into the analyzer. The stage plate 12 is mounted insidethe vacuum chamber for two dimension movement, e.g. by a conventionalstage mounting and stepper drives, allowing illumination of any desiredlocation of the sample or samples 2 on the substrate 3. Preferably, thesubstrate 3 has a somewhat conductive and dielectric thin layer ofmaterial on its bottom (fiber facing) side, which electrically contactsstage 12 and thus charges to an identical voltage as the stage, therebyproviding a flat electrical field at the emission surface of thesubstrate 3.

[0050] In another aspect, the invention features a process for nucleicacid sequencing by directly analyzing nucleic acids contained in apin-tool by MALDI mass spectrometry. In a preferred embodiment, thenucleic acid obtained from the sample is initially amplified. Forexample, a PCR reaction can be performed to “master” mix withoutaddition of the dideoxynucleotides (d/ddNTPs) or sequencing primers.Aliquots can then be isolated via a conjugation means and transferred,for example to a sequencing plate, where d/ddNTPs and primers can thenbe added to perform a sequencing reaction. Alternatively, the PCR can besplit between A, C, G, and T master mixes. Aliquots can then betransferred to a sequencing plate and sequencing primers added.

[0051] For example, 0.4-0.5 pmol of PCR product can be used in acycle-sequencing reaction using standard conditions, allowing each PCRto be used for 10 sequencing reactions (10x A, C, G, and T). Thesequencing reactions can be carried out in a volume of 10 μl containing5-6 pmol of 5′-labeled sequencing primer in a standard 384 microwellplate allowing up to 96 sequencing reactions (3360 bases at 35 bases perreaction). Alternatively, a 192 microwell plate approximately 5×5 cm ina 12×16 format can be used. This format allows up to 48 sequencingreactions to be carried out per well, resulting in 1680 bases per plate(at 35 bases per reaction). The format of the sequencing plate willdetermine the dimensions of the transfer agent (e.g., pin-tool).

[0052] A pin tool in a 4×4 array (FIG. 10) can be applied to the wellsof the sequencing plate and the sequencing products captured, e.g., onfunctionalized beads, which are attached to the tips of the pins (≧1pmol capacity). During the capture/incubation step, the pins can be keptin motion (vertical, 1-2 mm travel) to mix the sequencing reaction andincrease the efficiency of the capture.

[0053] Alternatively, the nucleic acid can be directly captured onto thepintool, for example, if an electrical field is applied, as shown inFIG. 14. When voltage is applied to the pintool, the nucleic acids areattracted towards the anode. This system also purifies nucleic acids,since uncharged molecules remain in solution and positively chargedmolecules are attracted to the cathode. For more specificity, thepin-tools (with or without voltage), can be modified to containpartially or fully single stranded oligonucleotides (e.g., about 5-12base pairs in length). Only complementary nucleic acid sequences (e.g.in solution) can then be specifically conjugated to the pins.

[0054] In yet a further embodiment, a PCR primer can be conjugated tothe tip of a pin-tool. PCR can be performed with the solid phase(pin-tool)-bound primer and a primer in solution, so that the PCRproduct becomes attached to the pin-tool. The pin-tool with theamplification product can then be removed from the reaction and analyzed(e.g., by mass spectrometry).

[0055] Examples of different pin conformations are shown in FIG. 11. Forexample, FIGS. 11A, 11B and 11C show a solid pin configuration. FIGS.11D and 11E show pins with a channel or hole through the center, forexample to accommodate an optic fibre for mass spectrometer detection.The pin can have a flat tip or any of a number of configurations,including nanowell, concave, convex, truncated conic or truncatedpyramidal (e.g. size 4-800μ across×100μ depth). In a preferredembodiment, the individual pins are about 5 mm in length and about 1 mmin diameter. The pins and mounting plate can be made of polystyrene(e.g. one-piece injection moulded). Polystyrene is an ideal material tobe functionalized and can be moulded with very high tolerances. The pinsin a pin-tool apparatus may be collapsible (e.g., controlled by ascissor-like mechanism), so that pins may be brought into closerproximity, reducing the overall size.

[0056] For detection by mass spectrometry, the pin-tool can be withdrawnand washed several times, for example in ammonium citrate, to conditionthe sample before addition of matrix. For example, the pins can simplybe dipped into matrix solution. The concentration of matrix can then beadjusted such that matrix solution only adheres to the very tip of thepin. Alternatively, the pin-tool can be inverted and the matrix solutionsprayed onto the tip of each pin by a microdrop device. Further, theproducts can be cleaved from the pins, for example into a nanowell on achip, prior to addition of matrix.

[0057] For analysis directly from the pins, a stainless steel “mask”probe can be fitted over the pins in one scheme (FIG. 12) which can thenbe installed in the mass spectrometer.

[0058] Two mass spectrometer geometries for accommodating the pin-toolapparatus are proposed in FIG. 13. The first accommodates solid pins. Ineffect, the laser ablates a layer of material from the surface of thecrystals, the resultant ions being accelerated and focused through theion optics. The second geometry accommodates fibre optic pins in whichthe samples are lasered from behind. In effect, the laser is focusedonto the pin-tool back plate and into a short optical fibre (about 100μm in diameter and about 7 mm length to include thickness of the backplate). This geometry requires the volatilized sample to go through thedepth of the matrix/bead mix, slowing and cooling down the ionsresulting in a type of delayed extraction which should actually increasethe resolution of the analysis.

[0059] The probe through which the pins are fitted can also be ofvarious geometries. For example, a large probe with multiple holes, onefor each pin, fitted over the pin-tool. The entire assembly istranslated in the X-Y axes in the mass spectrometer. Alternatively, as afixed probe with a single hole, which is large enough to give anadequate electric field, but small enough to fit between the pins. Thepin-tool is then translated in all three axes with each pin beingintroduced through the hole for sequential analyses. This format is moresuitable for the larger pin-tool (i.e., based on a standard 384 wellmicroplate format). The two probes described above, are both suitablefor the two mass spectrometer geometries described above.

[0060]FIG. 15 schematically depicts the steps involved in massspectrometry sequencing by post biology capture as described above.

[0061] This completes a description of basic embodiments of theinvention and representative constructions for implementing the improvedlaser desorption of the present invention. The invention being thusdisclosed and described, further variations and modifications will occurto those skilled in the art and all such variations and modificationsare considered to be within the scope of the invention, as defined bythe claims appended hereto.

What is claimed is:
 1. An improved method for matrix assisted laserdesorption/ionization (MALDI) of an analyte material, such methodcomprising the steps of preparing a specimen comprised of a majorportion of matrix material and a minor portion of analyte material,wherein said matrix and analyte are deposited as a sample on a supportilluminating said matrix such that material including the analyte isdesorbed from a surface of the sample and ionized, and initiallyconfining said desorbed material such that desorption momentum is atleast partially directionally randomized by collisional interactionthereby conditioning the desorbed analyte to control its initialconditions for analysis.
 2. The improved method of claim 1, furthercomprising the step of providing a collision buffer gas while confiningsaid desorbed material, said collision buffer gas being selected fromamong gases having a property to promote at least one of i) collisionaldissociation, ii) chemical ionization, and iii) molecule-molecule orion-molecule reaction with the analyte.
 3. The method of claim 1,wherein the step of illuminating includes the step of illuminating witha beam output from an optical fiber.
 4. The method of claim 3, whereinthe step of illuminating is effected by imaging the fiber output end onthe sample.
 5. The method of claim 3, wherein the step of illuminatingis effected directly by placing the fiber close to an opticallytransparent substrate carrying the sample and illuminating through thesubstrate.
 6. The method of claim 1, wherein the step of confining iseffected by confining with a baffle.
 7. The method of claim 1, whereinthe step of confining is effected by confining in a partially closedcompartment.
 8. The method of claim 1, wherein the step of confiningincludes permeating said desorbed material through a porous layer ofmaterial.
 9. The method of claim 8, wherein said porous layer ofmaterial is the substrate.
 10. The method of claim 1, wherein the stepof confining is performed by depositing said specimen as a permeable bedon the support and illuminating an underside of the bed so that desorbedmaterial is confined by the support.
 11. The method of claim 1, whereinthe step of confining includes confining with at least one electricpotential defining structure, said electric potential defining structurehaving a voltage selected to provide a residence time effective for saidions to initially thermalize by collisional interaction.
 12. The methodof claim 1, wherein the step of confining is effected by applying anextraction voltage only after a delay following desorption of saidmaterial thereby only extracting or sampling a portion of desorbedmaterial which has undergone collision.
 13. A method of sampling amatrix assisted laser desorbed ionized material comprising the steps ofilluminating a matrix to desorb material, and passing said materialthrough a permeable barrier effective to at least partially thermalizethe desorbed material thereby controlling initial conditions foranalysis of said material.
 14. Apparatus for providing an ionized sampleof material to an analysis instrument such as a mass spectrometer or thelike, the apparatus comprising a stage for holding a specimen from whichsaid sample is to be provided means directed at the stage forilluminating the specimen to cause desorption of material from thesample and ionization thereof, whereby the desorbed material has adirectional momentum, and means for confining said desorbed materialsufficiently to at least partially thermalize said directional momentumso that large molecules in said material are reduced in velocity,saidmeans for confining having an outlet communicating with the analysisinstrument to provide the large molecules of reduced velocity thereto.15. Apparatus according to claim 14, wherein the means directed at thestage includes an optical fiber spaced from said specimen to illuminatea defined region thereon.
 16. Apparatus according to claim 15, whereinsaid stage and said fiber are mounted for mutual relative motion toselectively scan the defined region of said specimen past anillumination position for sampling portions thereof.
 17. Apparatusaccording to claim 14, wherein the means for confining includes porousmaterial across a path to said outlet.
 18. Apparatus according to claim14, wherein the means for confining includes electrostatic means formaintaining desorbed material in a limited region to enhance collisionalinteraction before said outlet.
 19. Apparatus according to claim 14,wherein the stage contains a conductive region defining a flat fieldadjacent said specimen.
 20. Apparatus for matrix assisted laserdesorption of a large molecule analyte, such apparatus comprising asupport stage including means for holding a sample on a substrate in adefined region of the stage an optical fiber having a first end adaptedto couple to a light source for receiving a light input, and a secondend held by said stage in a position to illuminate a spot region on thesample, the stage positioning the second end so that its output beampasses through the substrate to directly illuminate said spot region onthe sample with a spot of defined size at a fluence effective to desorbthe analyte from the sample, whereby the desorbed material has adirectional momentum and the apparatus further comprises a means forconfining said desorbed material sufficiently to at least partiallythermalize said directional momentum.
 21. Apparatus according to claim20, comprising a plurality of optical fibers in an array arranged toilluminate an array of separate spot regions of the sample, each fiberbeing separately actuable to desorb material from a respective spotregion of the sample at a distinct time whereby plural samples ofdesorbed analyte may be provided at successive instants to a common massanalyzer.
 22. Apparatus according to claim 20, wherein the stage isoperative to move the substrate with the sample in at least two lateraldimensions relative to the second end of the optical fiber for aiming atarbitrary locations on the sample to desorb material.